Method of producing 5&#39;-nucleotide by phytopathogenic microorganisms



United States Patent 3,276,971 METHOD OF PRODUCING 5-NUCLEOTIDE BYPHYTOPATHOGENIC MICROORGANISMS Hiroshi Tone, Kawasaki, Ko Sasaki andYoshio Sayama,

Yokohama, and Tadashi Sakamoto, Tomoyuki Ishikura, and Noboru Miyachi,Tokyo, Japan, assignors to Sanraku Ocean Kabushiki Kaisha No Drawing.Filed Dec. 16, 1965, Ser. No. 514,403 Claims priority, applicationJapan, Jan. 20, 1962, 37/

1,862; Apr. 30, 1962, 37/ 16,780; May 1, 1962, 37/

17,361; June 11, 1962, 37/23,293

9 Claims. (Cl. 195-28) This application is a continuation-in-part ofSerial No. 248,203 filed December 31, 1962 and now abandoned.

The present invention relates to a method of producing highly purified5'-nucleotide with high yield from nucleic acid by using an enzyme orenzyme system with hitherto unknown properties which is formed by aphytopathogenic microbe; conducting the enzyme reaction at anunconventionally high temperature; and eliminating or minimizing theunnecessary by-products through said high temperature reactionand thepeculiar properties of said enzyme.

The nucleotide which is derived from the nucleic acid, has manyapplications other than academic. Especially the 5-nucleotide, thenucleotide with a phosphoric acid group in the 5'-position of ribose, isan important substance from a biochemical standpoint and is recentlybecoming important, particularly in the pharmaceutical and foodindustry.

Heretofore phytopathogenic microorganisms have not been applied in thefield of nucleic acid hydrolysis. It has now been found that several ofthese microorganisms possess enzymes or enzyme systems which are capableof hydrolyzing nucleic acid to form specifically 5 -nncleotide.

Enzymes or enzyme systems produced by phytopathogenie microorganisms aremade to react'wtih nucleic acid so that the latter is hydrolyzed to formspecifically 5'-nucleotides and the 5'-nucleotides thus produced arerecovered.

The microbes available for this method, as stated above, belong to genusMacrophomina, genus Ascoc'hyta, genus Phoma; for example, Macrophominaphaseoli (in Japan this is called Sclerotium bataticola) (ATCC No.14725), Ascochyta phaseolorum (ATCC No. 14728), Phoma cucurbitacearum)(ATCC No. 14864), etc. All of these are phytopathogenic microorganisms.

Some of the mycological nad phytopathogenic properties of these microbesare known, but their singular property of producing enzymes that canform specifically 5'- nucleotide has been unknown and the presentinventors are the first to reveal this property.

These microbes can be cultivated on the common cul- ,ture medium, thatis, using starch, sucrose, maltrose, glucose, etc., as the source ofcarbon, and nitrate, ammonium salt, peptone, etc., as the source ofnitrogen, but these being phytopathogenic microorganisms, it would hemore desirable to adopt the medium containing organic substances. Incase they are aerobic, either solid culture or liquid culture will beavailable. If bran is employed as the medium of solid culture noreplenishment of sugar The enzyme or enzyme system produced by themicrobes thus cultivated can be used in whatever form,

"ice

provided its activity is retained; for instance, a solid culture, itsextract; a liquid culture; its filtrate; a living cell, a dried one, ortheir grindings, lysates or extracts thereof;

or an enzyme preperation obtained by purifying through salting out,organic solvent precipitation, adsorption, chromatography or othermeans. As the starting material, polymerized nucleic acids, or partiallydegraded nucleic acids may be used. Both ribonucleic acid anddesoxyribonucleic acid may be used, 'but ribonucleic acid is moreeconomical. When it is extracted from a living cell or tissue, usuallythe extract can be employed without further purification.

The prominent features of this invention lie in the following points:

The 5'-phosphodiesterase produced by these strains has a remarkablyhiger optimum temperature than the hitherto known enzymes; accordinglythe hydrolysis reaction for obtaining 5'-nucleotide from ribonucleicacid (RNA) using this enzyme can take place at such high temperaturesabove 65 C. as C., and this high-temperature hydrolysis has variousadvantages as described later.

Chemical reactions, when conducted under high temperaures, are liable to'be attended with side reactions, which tend to diminish the yield oftarget substance or increase the impurities in it. If, however,hydrolysis by enzyme action is carried out at high temperatures, manyadvantages not obtained at low temperatures will be gained.

First, the enzyme can be utilized in its crude form. Microbes, whencultivated, will usually produce not simply one kind of enzyme.Therefore, if as is commonly done, a culture or its extract is directlyemployed as v the enzyme solution, enzymes present other than thenecessary one often will catalyze undesirable side reactions, the resultbeing a lowering of the yield of target substance or increasing theimpurities.

In the formation of 5'-nucleotide through reaction of5'-phosphodiesterase from RNA, enzymes which cause undesirable sidereactions are 3-phosphodiesterase (socalled ribonuclease, etc.) whichhydrolyzes RNA into 3'-nucleotide and phosphatase (including5-nucleotidase) which further hydrolyzes 5'-nucleotide, i.e., convertsthe target substance into nucleoside and inorganic phosphorus. Usually,even the microorganisms which have been isolated as5-phosphodiesterase-producing strain produce, though in smaller amountthan 5-phosphodiesterase, some enzymes which cause these undesirableside reactions. Therefore, -to inhibit such undesirable side reactionsdue to these enzymes, the conventional method has been to adopt suchenzyme inhibitors as sodium fluoride, phosphates, arsenates, cyanates,amoni acids, e.g. cystein, glutamine, ethylenediamine tetraacetic acid,metal ion, e.g., Z++ Cu++, etc. But additions of these agents are likelyto raise the production cost and also affect the process of separatingand refining 5-nucleotide from the reaction mixture. Particularly,suchadditions are unfavorable for the purpose of obtaining highlypurified 5- nucleotide, 'because of the possibility of contamination byimpurities.

Meanwhile, said side reaction-catalyzing enzymes can react at 30 C.40 C.but at higher temperature they will lose activity and not be able toreact. Therefore, if it is possible to hydrolyze RNA to 5-nucleotide atsuch high temperature as to inactivate the enzymes causing theundesirable side reaction, the addition of the said conventionalinhibitors will become needles-s; and the higher the reactiontemperature, the more perfectly the reactions can be inhibited. Thus,given the knowledge of the art prior hereto, it would be necessary topurify 5-phosphodiesterase to such extent that the ratio of purified5-phosphodiesterase to said side-reaction-catalyzing enzymev 75" C.,said side-reaction-catalyzing enzymes will be substantially completelyinactivated.

For this reason, if a strain that can produce such hightemperature-active enzyme is employed as source of 5' phosphodiesterase,the enzyme may be used even in its crude form.

Second, as stated before, .5'-nucleotide, i.e., the target product, caneasily be obtained with high purity and high yield. Namely, whensubstances other than 5-nucleotide, particularly 3-nucleotide, etc., areundesirably formed through side reactions, they must be separated from5'-nucleotide. This, industrially, presents a very difiicult job; itinvolves a risk of lowering the yield of 5'- nucleotide. That is, theformation of substances other than '5-nucleotide from RNA will mean alower yield of 5'-nucleotide.

Third, high reaction temperature greatly accelerates the reaction rateand this will prove very favorable for industrial production.

Experimental examples of these points are to be cited here.

Phoma cucurbitacearum (ATCC 14864) was cultured on bran medium and theextract therefrom was taken as an enzyme solution. Then thethermostabilities of the four enzymes contained in said solution,namely: 5'-phosphodiesterase, 3'-phosphodiesterase, phosphatase(5'-nucleotidase) and 3'-nucleotidase to be mentioned later wereinvestigated.

Said enzyme solution was treated for minutes at each temperature between20 C. and 100 C. and thereafter the residual enzyme activity wasmeasured with the results as follows:

Phospha- Temperature C. 5'-Phospho- 3-Pl1osphotase (5-3-Nucleodiesterase diesterase Nucleotidase tidase) Percent PercentPercent Percent As seen from above, 3'-phosphodiesterase andphosphatase, which catalyze the undesirable side reaction, completelylose their activities under high temperatures.

Reaction temperatures of these enzymes are as follows, with thetemperature at which maximum activity is exhibited being given as 100.

tion at 40 C. and that at 70 C., the yield of 5-nucleotide after thesame lapse of time is about 12 times as large .in the latter reaction asin the former, which means that the rate of latter reaction is about 12times as fast as that of the former.

As described above, in the formation of 5'-nucleotide through hydrolysisof RNA by 5'-phosphodiesterase, if 5-phosphodiesterase reacting between65 C.-75 C. is used, a great industrial advantage will emerge: Using acrude enzyme solution, highly purified 5'.-nucleotide can be obtainedwith high yield and in short time at that.

As explained above, elevation of reaction temperature in enzyme reactionoffers an industrial advantage; but the enzyme used for this purpose isitself protein and will be denatured and lose its activity when exposedto high temperature. This is a remarkably difierent property of enzymefrom common catalysts in chemical reactions.

Therefore, there is a limitation to theincrease in the temperature: Thetemperature shall not be so high as to denature the enzyme protein anddeprive it of its activity. Thus, it would be an extreme difficulty tofind any enzyme that can react at an exceedingly high temperature and insuch upper ranges; the higher the temperature, the more diflicult itwill be. Since common protein is completely denatured around 65 9 C., itwould be impossible firom the traditional conception of enzyme to thinkof any enzyme which can react at C.- C., i.e., 5 C.-10 C. higher thanthe above-mentioned temperature, without losing its activity, and whichfinds said range. as an optimum operating temperature. This would beeasy to understand in view of the tremendous efforts being rendered byenzymologists to prevent enzymes from being denatured and losing theiractivity through rise of temperature in their handling of enzymes.Hitherto it has been a common-sense practice in the conventionalhandling of enzymes to try to keep them 0001 in the course ofextracting, preserving or reacting them. From the fact that the optimumtemperature for 5'-phosphodiesterase is as high as 70 C.75 C., it isobvious that the 5'-phosphodiestenase, adopted in this invention is aunique enzyme protein beyond the scope of conventional conceptions ofenzymes and their action.

The 5'-phosphod-iesterase produced by the strains used in this inventionis a novel enzyme hitherto unknown which possesses the dollowingproperties and offers industrial advantages derived therefrom.

This 5-phosphod-iestenase has, as Well as 5'-'phospho diesteraseactivity, the 3-nucleotidase activity which, as

shown below, hydrolyzes 3'-nucleotide into nucleoside and inorganicphosphorus.

Action of. 5 -phosphodiesterase:

Action of 3'-nucleotidase:

covered by the present inventors, This discovery has been reported toacademic circles by the present inventors. For example, 5-phosphodiesterase which was isolated and purified from the extract ofPhornla cucurbitacearum culture was confirmed to be homogeneous byultra-centrifuge sedimentation, electrophoresis, and chromatography; its5-phosphodiesterase activity and 3-niioleotidase activity behavedentirely the same; and the thermal inac- 5 fact that, as described aboveand proved experimentally, tivation land the optimum reactiontempenature were also the optimum temperature is within the rangebetween the same for both these activities. Hydrolysis of RNA 65 C.7S C.for both 5'-phosphodiesterase activity and into 5'-nucleotide by meansof such unique 5'-phospho:di- 3'-n-ucleotidase activity; if thattemperature were lower esterase possessing both 5-phosphodiesterase and3-nufor either of these activities, the earlier-mentioned sidecleotidase activities offers the following industrial :advan- 10reaction might happen, but as stated above, both act at tage. the samehigh temperatures, so that there is no possibility The starting materialof 5-nucleotide production, i.e., of side reaction. High reactiontemperature of both en- RNA is usually extracted from yeast; when it isinduszyme activities will also prove favorable in the following triallyextracted, hydnolysis will occur in the course of case. extraction andinconsequence the polymerization degree In the industrial hydrolysis ofRNA, when a troublewill fall; through this hydrolysis, phosphorusremains in occurs in the temperature control or in the preparation the3'-end group of the extracted RNA. of reacting solution and as theresult the reacting tem- X Y z X Y z Extraction X Y X Y As thepolymerization degree decreases, the end group perature fails to go soas to reach within the range bewith phosphorus bonded at 3-position willincrease. tween 65 C.7S C., 3-nucleotide which may be formed When thisRNA is hydrolyzed by 5'-phosphodiesterase, through the undesirable sidereaction will be transformed 5-nucleotide will be produced and at thesame time nuinto nucleoside through the action of 3'-nucleotidase bycleoside 3',5-diphosphate will be formed from the :aboverestoring thereacting temperature to the normal range; mentioned end group withphosphorus bonded lat 3'-pomoreover, separation of nucleoside fromnucleotide will sition. be easy, so that there is absolutely no fear of3'-nucleotide X Y Z infiltration into 5'-nucleotide. 5' 3' 5 3' 5' 3Thus, the possible loss in the yield of 5'-nucleotide through a troublein its industrial production can be held to a minimum and highlypurified 5-nucleotide can be recovered. g g 40 As described above, thepresent invention relates to a P P method of obtaining highly purified5-nucleotide at high Formation of nucleoside 3,5-diphosphate in thismanyield y Y Y g RNA y a unique enlyme, Said n mean that the will notTecover as 5'- enzyme being produced from Phoma cucurbitacearum,nucleotide and lead to a poor yield of 5-nucleotide Ascochytflphaseoloi'um M acl'ophor phaieoli and When in this way nucleoside3',5'-diphosiphate is chracteflled y possesslng b 5'-phbspbodlesteraseformed in the reaction mixture, there is a likelihood of not! and3"m1c1eot1da actlon Optlmum ltem' nucleoside 3,5-di'ph-oisphateinfiltering into 5 -nucleotide peraturfis enZymb tlvityare as high as 65C.75 in the separation and purification of 5'-riucleotide from Forlsolatlon and Purlficatlon 0f the p b 501m? the reacting solution; thiswould be a serious disadvantage q p between the fQrmed a the for theproduction of highly purified 5-iiucleotide. And gurltles to be remoYed1n physlqfll, qhcmlcal P thi-s disadvantage would be the more serious,when RNA i165 l as adsorptlbn, b lf, lf P P PY, employed as Startingmatch-a1 is the lower in l i are utilized and the 5 -nucleotide isobtained in an isolated mm degree form or in the form of salts. Anionexchange resin is To liquidate such loss, the phosphorus at the end hasrecommendable for individual separation of the four kinds only to behydrolyzed preliminarily; but such hydrolysis of 5 q would be chemicallyhard to execute, moreover such by. Th tollowin eXamplbS b P P 0f dmlysisin the course of enzyme reaction would raise the illustration only anddo not lunit the invention thereto.

rodu-ction cost. p When, however, RNA is hydrolyzed using the enzymeExample I as obtained from the strainsused in this invention which phcucwbimceamm (ATCC 148 4 i l fro P08565565 both 5''Pbosphodlestbl'aseymucleotldase Cucurbita moschata var. toonas was inoculated on 300 g.activity, the I' Q P the 3"te1'm1na1 of the f of culture mediumconsisting of one part of bran and one erized RNA will be previouslyhydrolyzed to inorganic part of Water; after 10 days at C, it wasextracted phosphorous by the actionof 3 -nucleot1dase and then the with750 m1. of water To 700 of the extract was i.ephoSphOry1at.ed W111 behydrolyzed g added 30 g. of yeast ribonucleic acid dissolved in about pby the aCt.10n of 5 P F F Wu out 1.0 liter of water. The resultingmixture was adjusted ing the undesirable nucleotide 3 ,5 -diphosphate.And t 0 t M t t 1 1 f3 0 also the nucleoside 3',5'-diphosphate formedfrom the 9 ace 10 f 0 yle a v0 ume.o 3, termina1 of the depolymefized RNA by the action of 111601. T en it was su ected to two-hour reaction at5-phosphodiesterase may be transformed into 5'-nucleo- 70 and zo'mmutesheat treatment at u and tide by the simultaneous action of3'-nucleotidase. Anythereafter was filteredfiltrafe was adlusted way,simultaneous action of both these activities will be P by 4 and 5nucleotlde adsorbed on able to yield 5-nucleotide with no residue ofnucleoside DOWeX 1 -WP AS the result of 6111mm y M/SOO-3',5'-diphosphoric acid. Thus, without making any spe- M/ 100 HCl, 5CMP3.82 g. 5-AMP 7.94 g., 5'-UMP cial treatment and causing any rise in thecost, the loss 7 7.03 g. and 5'-GMP 5.79 g. were obtained.

can be prevented. Accordingly, even if the quality of starting material,i.e., RNA is inferior, 5'-nucleotide will be obtained with high purityand maximum yield.

Meanwhile, an additional advantage is provided by the 7 Example 2Ascochyta phaseolorum (ATCC 14728) was inoculated on 300. g. of culturemedium consisting of one part of bran and one part of water; after 10days at 25C., it was extracted with 750 ml. of water. To 700 ml. of theextract was added 30 g. of yeast ribonucleic acid dissolved in about 1.0liter of water. The resulting mixture was adjusted to pH=5.0 with aceticacid to yield a total volume of 3.0 liter. Then it was subjected totwo-hour reaction at 70 C. and 20-minute heat treatment at 100 C.; andthereafter was filtered. The filtrate was adjusted to pH=9.0 by NH OH;and 5'-nucle0tide was adsorbed on Dowex 1 (Cl-type). As the result ofelution by M/500- M/ 100 HCl, 5'-CMP 3.60 g., 5'-AMP 7.60 g., 5-UMP 6.80g., and 5-GMP 5.58 g. were obtained.

Example 3 Using Macrophomina phaseoli (ATCC 14725) causing gray stem rotin Vingner sinensis, 5-CMP 3.55 'g., 5-AMP 7.66 g., 5-UMP 6.92 g. and5'-GM-P 5.62 g. were obtained in the same as in Example 1.

Example 4 A culture medium was made from glucose 5%, NH C1 0.3%, peptone0.5%, yeast extract 0.95%, KH PO 0.05%, K HP 0.05%, CaCl -2H O 0.04%,

0.04% (pH=6.0). from Cucurbita moschata var. toonas was inoculated in500 ml. of this solution of which 50 ml. had been placed in a 500 ml.shaking flask. This was incubated with shaking at 25 C. After sevendays, the grown cells were harvested by filtration, washed with a cold0.9% NaCl solution, ground together with a small amount of a M/20 trisbufier solution (pH=7.0) and quartz sand,-and then centrifuged.

Fifty m1. of the cell-free exttract thus prepared was mixed with 25 ml.of 3% yeast ribonucleic acid solution and 25 ml. of a M/3 acetate buffersolution (pH=5.0) to yield 150 ml. in total volume. After two hoursreaction at 70C., 50 ml. of uranyl reagent was added; the precipitatewas removed by centrifugation after cooling, and the supernatantobtained was neutralized with KOH. The quantity of -nucleotide containedin the resultant solution was determined, using a Dowex I (formate type)column; the yields were 5'-CMP 105 mg., 5'-AMP 220 mg, 5'-UMP 197 mg,and 5'-GMP 161 mg.

What is claimed is: 1. A method for producing 5'-nucleotides, whichcomprises: (a) culturing in a culture medium at a tempera- Phomacucurbitacearum isolated ture of 18 C. to 33 C., inclusive, a strainselected from the group of microorganisms consisting of genus Phoma,genus Ascochyta and genus Macrophomina, which thereby produces anenzymatic active substance capable of forming specifically 5-nucleotidefrom nucleic acid, said enzymatic active substance possessing both5-phospho'diesterase and 3'-nucleotidase enzymatic activity, (b)separting said enzymatic active substance from the micro-. organismculture, (c) reacting said enzymatic active substance at a temperatureabove C. and as high as C., with nucleic acid solution having an acidpH, thereby forming the 5-nucleotides, and (d) recovering the thusformed 5-nucleotides.

2. A method as claimed in claim 1 in which the culture medium is a solidmedium and the strain is cultivated at a temperature of 25 C. to 30 C.

3. A method as claimed in claim 1 in which the culture medium is aliquid medium and the strain is cultivated at a temperature of 25 C. to30 C.

4. A method as claimed in claim 1 wherein the enzyme is reacted with thenucleic acid solution at a temperature above 65 C. and as high as 75 C.,inclusive, said solution having a pH of about 5.0.

5. A method as claimed in claim 1 in which the strain which producessaid enzymatic active substance is a strain selected from the groupconsisting of Phoma cucurbitacearum (ATCC 14864), Ascochyta phaseolorum(ATCC 14728) and Macrophomina phaseoli (ATCC 14725).

6. A method as claimed in claim 1 in which said enzymatic activesubstance contained in said culture is separated from at least onesubstance selected from the group consisting of the culture extract, theculture filtrate, the living cells, the dried cells, their lysate, andcell free. extract.

7. A method as claimed in claim 1 which the culture medium is a solidculture medium and comprises bran as its main constituent.

8. A method as claimed in claim 1 in which said enzymatic activesubstance is in crude form;

9. A method as claimed in claim 1 in which said enzy? matic activesubstance is purified.

References Cited by the Examiner UNITED STATES PATENTS 9/1963 Sakaguchiet al. 2/1964 Tanaka et al.

1. A METHOD FOR PRODUCING 5''-NUCLEOTIDES, WHICH COMPRISES: (A)CULTURING IN A CULTURE MEDIUM AT A TEMPERATURE OF 18*C. TO 33*C.,INCLUSIVE, A STRAIN SELECTED FROM THE GROUP OF MICROORGANISM CONSISTINGOF GENUS PHOMA, GENUS ASCOCHYTA AND GENUS MACROPHOMINA, WHICH THEREBYPRODUCES AN ENZYMATIC ACTIVE SUBSTANCE CAPABLE OF FORMING SPECIFICALLY5''-NUCLEOTIDE FROM NUCLEIC ACID, SAID ENZYMATIC ACTIVE SUBSTANCEPOSSESSING BOTH 5''-PHOSPHODIESTERASE AND 3''-NUCLEOTIDASE ENZYMATICACTIVITY, (B) SEPARTING AND ENZYMATIC ACTIVE SUBSTANCE FROM THEMICROORGANISM CULTURE, (C) REACTING SAID ENZYMATIC ACTIVE SUBSTANCE AT ATEMPERATURE ABOVE 65*C. AND AS HIGH AS 75* C., WITH NUCLEIC ACIDSOLUTION HAVING AN ACID PH, THEREBY FORMING THE 5''-NUCLEOTIDES, AND (D)RECOVERING THE THUS FORMED 5''-NUCLEOTIDES.